Genetic analysis of cell cycle control in yeast
Introduction Model organisms are used for research because they provide a framework on which to develop and optimize methods that facilitate and standardize analysis. The yeast Saccharomyces cerevisiae is one of most widely used eukaryotic model organisms. It has been used as a model to study aging, regulation of gene expression, signal transduction, cell cycle, metabolism, apoptosis and many other biological processes(1). S. cerevisiae ''lend well to the study of cell cycle because it is easy to grow in culture and it has a short generation time, around 2 hours at 30C. Cell division cycle mutants isolated from S. cerevisiae allowed the identification of a variety of functions necessary for cell cycle progression. Mutations in the cell division cycle genes, called cdcs affect the cell at specific points in the cell cycle. These mutations block progression through the cycle without blocking general cell growth and macromolecular synthesis. Cell division cycle S. cerevisiae cell cycle begins with the initiation of bud formation, followed by DNA replication and nuclear division. Bud enlargement continues throughout the cell cycle until the bud reaches approximately the size of the parent cell at the time of cell separation. The process is terminated by cell separation. In culture rapidly growing yeast can be seen to have buds while in cultures growing more slowly because of depletion of nutrients or non-permissive temperatures, the cells lack buds and their formation is observed only during part of the cell cycle. The genetic control of the cell division cycle in S. cerevisae can be studied utilizing temperature-sensitive mutants. Hartwell used a large collection of temperature-sensitive mutants that arrested division at different points. He was the first one to identify many cdc genes that when mutated arrested the cell cycle division at different points. In those experiments cells from an asynchronous population first arrested normal development at the same point and nearly all of the mutant cells assumed the same aberrant morphology after extended incubation at the restrictive temperature. (2) Many cdc genes have been identified to date, each one is involved in a different part of the cell cycle. Method Strains used: -Parent strain A364 - Derived haploid temperature-sensitive mutants: ts-369, ts-370, and ts-104. These strains were obtained by mutagenesis with nitrosoguanidine. The diploids were obtained by crossing each of these haploids to a nontemperature-sensitive strain, 79-20-3 the resulting diploid was allowed to sporulate and tetrads were dissected by micromanipulation. Temperature-sensitive haploids of opposite mating type segregating from this cross were in turn mated to obtain diploids homozygous for the temperature sensitive lesion; the temperature-sensitive diploids were designated 369 D-1, 370 D-1, and the mutations were cdc-1, cdc-2, and cdc-3 respectively. Time-lapse photomicroscopy was utilized to detect temperature-sensitive yeast mutants that were defective in gene functions needed at specific stages of the cell-division cycle. The technique provided information about the time at which the defective gene function was normally performed, defined as the execution point, and the stage at which the function was not performed, defined as the termination point. The cells were grown for several generations at the permissive temperature of 23 C in YM-1 medium and then collected by centrifugation while still in the exponential growth phase. Cells were resuspended at a density of 2x10^7 cells/ml and spotted onto a YEPD plate pre-warmed to the restrictive temperature 36C. Photographs were taken within 10 min after spotting, and at various times thereafter. Results Visual observation of the relative size of the bud and parent cell revealed the point to which a particular cell had progressed through the cycle. Three mutants carrying three different genes were identified: 369 D1, 370 D1 and 104 D1 carrying the genes, cdc-1, cdc-2, and cdc-3 respectively. The genes were involved early in the cell cycle, at about the time of bud initiation, but differed in their termination points. Cells carrying the cdc-1 mutation terminated at the execution point, most cells ended up with a tiny bud that did not develop further. Cells carrying the cdc-2 mutation terminated at mitosis, cells with buds (small or large) at the time of the temperature shift continued through the cell cycle but did not seperate. Doublets and quadruplets were observed but no small cells. Cells carrying the cdc-3 mutation (figure 2D) were defective in cell separation and showed no definite termination point since other processes of the cell cycle, such as bud initiation and nuclear division, continued despite the stop in cell separation (3). Conclusions S. ''cerevisiae was used as a model organism to study cell cycle regulation. Using temperature sensitive yeast mutants Hartwell demonstrated that three genes: cdc-1, cdc-2, and cdc-3, were involved early in the cycle, at the time of bud initiation but differed in their termination points. Yeast carrying the cdc-1 mutation ended up with a small bud that did not develop further. Cdc-2 mutants terminated at mitosis, and cdc-3 mutants were defective in cell separation and showed no definite termination point. References 1. Karathia H, et al. Saccharomyces cerevisiae as a Model Organism:A Comparative Study. PLoS One. 2011; 6(2): e16015. Published online Feb2, 2011. doi: 10.1371/journal.pone.0016015 .PMCID: PMC3032731 2.Hartwell L et al. Genetic Control of the Cell-Division Cycle in Yeast.Detection of Mutants.I. Proc Nat Acad Sci. 1970. Vol. 66, No. 2, pp. 352-359. 3. Hartwell L. ''Saccharomyces cerevisiae. '' Cell Cycle. Bact.Reviews. 1974. Vol 38, No 2, pp 164-198.